Augmented Survival of Bacteria Within Biofilms to Exposure to an Atmospheric Pressure Non-Thermal Plasma Source
Bacteria embedded within biofilms present a challenge to surface decontamination by conventional means. Atmospheric pressure non-thermal plasma processes have emerged as a promising approach to overcome this problem. We used a non-thermal atmospheric pressure plasma, operated in a humid atmosphere, to assess planctonic versus biofilm-resident bacterial (Escherichia coli) susceptibility to treatment. The concentrations of stable chemical species at the treatment reactor gas outlet were monitored by FTIR. The decontamination efficiency of the process was evaluated against bacteria embedded within a biofilm, as well as planctonic cells placed on a glass surface. Bacterial survival was assessed using a combination of Colony Forming Unit (CFU) ability and vital staining with a combination of DAPI plus Propidium Iodide. Both methods revealed an increased resistance of biofilm-resident bacteria, when compared to planctonic cells, after a 40 min exposure to the post discharge gas. Present results show that biofilm-resident bacteria demonstrate augmented survival when exposed to atmospheric pressure non-thermal plasma treatment and thus that decontamination procedures should take into account this survival when evaluating surface decontamination measures.
Biofilms consist of microorganisms embedded in biological polymers which can
be composed of polysaccharides, proteins and DNA. They form complex structures
notorious for their resistance to various treatments such as antibiotics or
chemical/physical decontamination procedures. The exact mechanisms responsible
for their resistance are, as yet, not fully understood, though the expolymeric
substances (EPS) surrounding the cells can act as a physical/chemical protective
barrier (Odic et al., 2002; OToole
et al., 2000). Biofilm construction starts with bacterial cells adhering
to a surface. Microcolonies then develop that become increasingly complex in
structure and eventually shed cells that can act as founders for new biofilms
(Cho et al., 2007; OToole
et al., 2000; Patel, 2005). Bacteria within
biofilms are noted to be difficult to completely remove or disinfect from a
surface using known decontamination procedures (Kamgang
et al., 2007; Nadakumar et al., 2004).
Surface treatment methods, such as ultraviolet (UV) or X-ray irradiation, chemical
cleaning, heat treatment (with or without chemical treatment), present various
drawbacks, including high costs, inadequacy of complete bacterial inactivation
and material damage to fragile and/or complex surfaces such as electronic components,
catheters and other medical devices (Laroussi and Leipold,
2004). A serious need for research in this area is thus emerging.
Non-thermal plasma present a promising avenue for surface decontamination (Odic
et al., 2002; Pointu et al., 2008).
Non-thermal atmospheric pressure plasma processes are simple to implement and
yield, at moderate temperatures (generally room temperature), reactive chemical
species. The concentration and nature of these species (including free radicals,
excited molecules and stable species such as ozone) can be controlled, depending
upon the gas mixture and electrical parameters used. Light emission is also
produced by non-thermal plasma and according to the surface exposure mode, the
resultant UV emissions can contribute to surface treatment. Non-thermal plasma
have been widely studied to treat surfaces for a variety of purposes such as
polymer surface modification (Arefi-Khonsari et al.,
2005) and wafer surface cleaning for electronic applications (Radio-frequency
plasma) (Korner et al., 1995). Atmospheric pressure
non-thermal plasma processes have been shown to be effective for surface decontamination
of various agents, such as bacterial cells (Dodet et al,
2006; Kamgang et al., 2007; Kirkpatrick
et al., 2007; Moreau et al., 2005)
or prions (Baxter et al., 2005). It has also
been shown that non-thermal plasma processes can irreversibly damage bacterial
spores so that they are unable to grow and form colonies on Petri dishes (Odic
et al., 2002). For the present study, an atmospheric pressure non-thermal
plasma device was specifically developed for the treatment of biofilms present
on surfaces. This device presents the following advantages: operation at atmospheric
pressure, low investment (relatively simple device) and low energy costs (discharge
input power <2 W), minor heating of the gas and treated surfaces, a post
discharge exposure mode implying homogeneous treatment of the surface, adjustable
geometry of the plasma generation electrodes and thus variable surface area
treatment. For this study, the plasma device was operated in a humid atmosphere.
The decontamination efficiency of the process was evaluated using differential
staining of Escherichia coli bacteria directly within a biofilm, as well
as planctonic cells placed on a glass surface. Present results show that biofilm-resident
bacteria demonstrate greater survival to atmospheric pressure non-thermal plasma
treatment than planctonic cells and thus that decontamination procedures should
take into account this survival when evaluating surface decontamination measures.
MATERIALS AND METHODS
Strains and growth medium: Escherichia coli strain DH10B (F-,
mcrA, Δ(mrr-hsdRMS-mcrBC), φ80dlacZΔM15,
ΔlacX74, deoR, recA1, relA1, endA1,
araD139, Δ(ara, leu)7697, galU, galK16, galE15,
rpsL, nupG, spoT) (Durfee et al., 2008)
was stored at -20°C in glycerol (20% v/v). Bacteria were cultured at 30°C
in M63 minimal medium supplemented with glucose (0.2%) without agitation for
all experiments (Miller, 1972).
Planctonic cell preparation: The cells of a stationary phase bacterial culture were collected by centrifugation (4000 x g, 10 min, 5°C) and the cells re-suspended in water. After dilution, 10 μL of a 108 CFU mL-1 suspension were spotted on sterile glass microscope covers slips (15x15 mm, Menzel Glaser, Braunschweig, Germany) and subsequently dried at room temperature for 45 min prior to atmospheric pressure non-thermal plasma treatment.
Biofilm formation on glass coverslips: Sterile glass microscope covers
slips were placed in a 60 mm diameter plastic Petri dish containing 15 mL of
M 63 medium (Miller, 1972). Then, 200 μL of stationary
phase bacterial culture were added and the Petri dish incubated for a further
48 h at 37°C without agitation. The glass cover slips were then removed,
washed with distilled water and dried at room temperature for 45 min prior to
atmospheric pressure non-thermal plasma treatment.
Plasma treatment: The non-thermal plasma apparatus (Fig.
1) consists of two parallel insulated electrodes separated by a 1 mm gas
gap g as the electrical discharge cell. Each insulated electrode is a borosilicate
tube (external diameter 2xr = 6 mm, 1.5 mm thickness, 70 mm length) with its
inner surface partially covered with silver paste acting as an electrode (the
active length of the electrode is limited to 50 mm). One electrode is connected
to ground through a measurement resistor Rm and the other is energized
by a high voltage power supply (AC 28 kHz; 0-10 kVpeak). Micro filamentary
discharges develop in the gas gap g between the two parallel electrodes along
the 50 mm active length.
||Diagram of the (a) discharge cell and (b) plasma treatment
Arcing is prevented by the borosilicate insulators (relative permitivity εr
= 4.5) through a dielectric surface charge build-up mechanism leading to a decrease
of the local electrical field. Voltage and discharge current measurements were
made using a LeCroy PPE20kV 1:1000 voltage probe (Lecroy France, Courtaboeuf,
France) and the Rm 25 Ω resistor, respectively. Electrical signals
were recorded using a Tektronix TDS 544A 500 MHz oscilloscope (Tektronix S.A.,
Les Ulis, France). The mean discharge input power P was calculated by the instantaneous
voltage current product method integrated over two periods of the applied voltage
signal. The distance d between the dielectric tube arrangement and the surface
to be treated was fixed at 3 mm (Fig. 1a) and the plasma zone
was then located approximately 6 mm above the sample surface. The feed gas was
an air-like mixture (N2/O2-80/20) saturated, by bubbling
distilled water through a gas sparging bottle, with water vapor at room temperature.
The feed gas is homogeneously distributed in the electrode gap along the active
zone (50 mm) using a grid device (Fig. 1b). Two glass cover
slips, containing either planctonic or biofilm-resident bacteria, were simultaneously
exposed to the discharge effluents. This arrangement allowed the entire surface
of the glass cover slips to be exposed to the plasma effluent. The sample holder
(microscope slide covering a brass plate) was cooled to enhance water condensation.
The entire device, including sample holder discharge cell and feed gas injector,
was embedded in a treatment reactor. The gas outlet composition was monitored
using a Bruker FTIR absorption spectrophotometer (Bruker Optics, Champs sur
Marne, France). The experimental spectra were acquired using a feed gas consisting
of air at atmospheric pressure and ambient temperature. In order to subtract
overlapping peaks of water vapor absorption, the reference transmittance spectrum
was acquired in atmospheric air saturated (RH≥95%) at 50°C with water
For each treatment, four glass cover slips with the biofilm or planctonic cells
were prepared. One was kept at room temperature as a non-exposed control, while
the other two were placed on a microscope slide for plasma treatment. The slide
was then placed into the treatment zone. Typical operating conditions were as
||Exposure duration (treatment time): 40 min
||Mean discharge input power P: 1.7-1.8 W
||Gas flow rate: 2 NL min-1 (N2/O2-80/20,
Viability assays: After treatment, one of the cover slips was used for
determining cell viability by Colony Forming Unit (CFU). Using a sterile glass
microscope slide, the bacteria in the biofilm, or deposited planctonic cells,
were scraped from the cover slips into a 60 mm Petri dish containing 5 mL of
LB media (Miller, 1972) and then dilutions were spread
on LB agar. The colonies were then counted after a 30 h incubation at 30°C.
The other cover slip was stained and examined using DAPI (10 μg mL-1)
plus propidium iodide (5 μg mL-1), as previously described by
Doolittle et al. (1996), Jenkins
et al. (1997) and Lecoeur (2002).
The cover slips were then placed in the dark for 15 min prior to observation
by fluorescence microscopy.
In order to determine the relative sensitivity of biofilm-located versus planctonic E. coli cells to an atmospheric pressure non-thermal plasma source (post-discharge exposure mode), treatments were carried out using remote exposure of cover slip surfaces, containing the bacteria, to the effluent gas. A sketch of the experimental set-up and the plasma source are presented in Fig. 1. The plasma produced in the transient micro-discharges does not reach thermodynamic equilibrium (non-thermal plasma) and thus the bulk gas temperature remains close to ambient (data not shown). The gaseous feed mixture, streaming through the gas gap at atmospheric pressure, was activated by the discharge. The resultant non-thermal plasma-activated gas mixture was then allowed to flow over the sample surface. Thus, there was no direct interaction between the plasma and the samples. The non-thermal plasma-activated gas mixture which flowed over the sample surface was characterized using absorption spectroscopy (Fig. 2a). Corresponding stable species are shown along with the detected concentrations expressed in ppm (Fig. 2b). The negative absorption bands observed in the spectrum are due to (1) the CO2 (2300-2380 cm-1) absence in the non-thermal plasma-activated gas mixture and (2) its lower water vapor content (1350-1900 cm-1) when compared with the reference spectrum.
In order to test the efficacy of the non-thermal plasma-activated gas mixture
to inactivate bacteria, E. coli samples were prepared as either planctonic
cells or as cells embedded within a biofilm. Planctonic E. coli cells
were grown and subsequently deposited on sterile glass cover slips. Bacteria
in the stationary phase of growth were used because they are known to be more
resistant to chemical treatment (Dodd et al., 2007).
The viability of the bacteria before and after non-thermal plasma-activated
gas mixture exposure was determined by scraping bacteria from the cover slip,
followed by re-suspension, spreading and colony growth on LB agar. In addition,
cells were stained with DAPI and Propidium Iodide (PI) followed by direct fluorescence
||FTIR absorption spectrum of the treatment reactor gas outlet
performed at ambient temperature and atmospheric pressure. (a) The spectra
and (b) Stable gaseous oxidative species at the plasma reactor outlet were
||Fluorescence microscopic examination of cells prior to and
after exposure to an atmospheric pressure non-thermal plasma. (a) Stained
planctonic cells after 40 min exposure to ambient air (control); (b) Stained
planctonic cells after a 40 min non-thermal plasma treatment; (c) Stained
biofilm cells after 40 min exposure to ambient air (control) and (d) Stained
biofilm cells after a 40 min non-thermal plasma treatment. The corresponding
CFU mL-1 results for the recovered E. coli planctonic
cell and biofilm samples are displayed under each photograph
Propidium Iodide (PI) will only enter and stain DNA in membrane-damaged bacteria
and thus cells which are heavily damaged will appear red, while intact cells
will appear blue (Jenkins et al., 1997). Prior
to exposure to the non-thermal plasma-activated gas mixture, the vast majority
of cells stained blue (Fig. 3a), while after exposure all
cells stained red (Fig. 3b). These results were supported
by those using colony formation to asses cell viability, as no colonies were
observable after non-thermal plasma-activated gas mixture exposure of planctonic
cells, while the initial cell population was 4x106 CFU mL-1
prior to exposure (Fig. 3a, b).
Similar experiments were performed to determine the sensitivity of E. coli
cells embedded within biofilms on glass cover slips to non-thermal plasma-activated
gas mixture exposure. Scraping of cells from the cover slips, followed by re-suspension
and growth on LB agar, revealed an approximate 40 fold reduction of apparent
cell viability for the non-thermal plasma-activated gas mixture treated sample
compared to unexposed controls. Fluorescence microscopic examination, after
staining the biofilms with PI plus DAPI (Fig. 3c, d),
showed that approximately 50% of cells stained red following non-thermal plasma-activated
gas mixture exposure (Fig. 3d), whereas after a 40 min exposure
to ambient air only a relatively minor fraction of the cells were found to stain
red (Fig. 3c).
The decontamination of fragile material, such as plastics, has led to the search
for alternative treatments to those (e.g., wet or dry heat) currently in use.
As an example, atmospheric pressure ozonizers have been used to prevent microorganisms
from proliferating in drinking water (Kogelschatz, 2000).
Large quantities of ozone are required (in the range of 10,000 ppm) which, if
utilized for surface treatment, could affect the integrity of fragile polymeric
materials. Moreover, the recognition of the ubiquity of bacterial biofilms has
also added to the necessity to discover efficient bacterial inactivation processes.
Bacteria within biofilms are known to be more resistant to chemical and physical
treatments (Nadakumar et al., 2004; Patel,
2005), with the comcomitant necessity for the use of alternative procedures
for their removal and/or inactivation. One of these alternatives is the use
of electrically-generated plasma processes as a surface decontamination technique
(Laroussi et al., 2002; Moreau
et al., 2005). These processes can include direct exposure to thermal
or non-thermally generated plasma, but may also comprise exposure of the test
surface to reactive chemical species generated in a gas presented to the plasma
The use of an atmospheric pressure non-thermal plasma process presents several advantages for surface contamination over thermally-generated plasma, including the lack of heating the substrate. In addition, there is no direct interaction between the electrically-generated plasma and the treated sample. Thus, surface decontamination is caused by exposure of the surface to be treated to short-lived chemical species generated in the surrounding gas by the electrically-generated plasma processes. Only the active chemical species produced by the plasma are transferred to the surface, thus ensuring a generally homogeneous treatment of the sample. Additionally, the active treatment chemical species can be controlled by the initial composition of the input gas and the electrical parameters of the plasma discharge.
In this study, wet air was used as the feed gas, as it is readily available.
Under our conditions, a water film was formed on the surface during the treatment
through condensation of water from the humid air used as a feed gas. The water
film pH was found to decrease from pH 6 to 1.8 at the end of the treatment (data
not shown). In earlier studies using atmospheric pressure non-thermal plasma
processes, the pH of aqueous samples was found to decrease to values ranging
from pH 2 to 4, depending on experimental conditions (Kirkpatrick
et al., 2007; Moreau et al., 2005;
Odic et al., 2002; Pointu
et al., 2008). It is thus possible that a decrease in pH played a
role in cell inactivation, as E. coli is known to be sensitive to acidic
pH conditions (Goodson and Rowbury, 1989).
The stable oxidative species, such as ozone (O3), nitrogen oxides
(mainly N2O and NO2) and the associated nitric acid, HNO3,
were identified and quantified. During the entire treatment, an average ozone
concentration of 175 ppm was measured at the reactor outlet. Singlet oxygen
O2 (1Δg) biocidal activity has been shown
to act as a membrane-disorganizing agent in Staphyloccocus aureus and
as a DNA-damaging agent (Maisch et al., 2007;
Ravanat et al., 2000). The OH and HO2
radicals produced by means of an atmospheric pressure wet argon non-thermal
plasma were found to be responsible for the inactivation of E. coli cells
by 5 orders of magnitude within 20 min (Dodet et al.,
2006). More generally, reactive oxygen species are known to be efficient
at killing microorganisms (Waris and Ahsan, 2006). Thus,
a combination of a decrease in pH and the presence of Reactive Oxygen Species
(ROS) coming from the activated gas phase are likely to play important, though
not necessarily exclusive, roles in cell morbidity and mortality casued by non-thermal
atmospheric pressure plasma processes (Goodson and Rowbury,
In the present study, fluorescence microscopy examination of in situ
stained cells, in addition to measurement of viable cells through CFU formation,
were used to assess cell viability prior to and after non-thermal atmospheric
pressure plasma treatment. We found that planctonic E. coli cells were
very sensitive to treatment, with a significant loss of viability measured by
both assays, as we observed a marked diminution of CFU ability and a marked
uptake of propidium iodide by cells after treatment. This latter observation
is in agreement with the results of Laroussi et al.
(2002), who observed, using Scanning Electron Microscopy (SEM), that non-thermal
atmospheric pressure plasma processes could disorganize E. coli cell
membranes. In addition, planctonic Erwinia sp. were also found to be
sensitive to a similar plasma activated gas mixture (Moreau
et al., 2005). In contrast, biofilm-resident cells appeared to be
more resistant than planctonic cells to exposure to the plasma activated gas
mixture, a result in concordance with those obtained using a gliding discharge
plasma treatment in humid air with Staphylococcus epidermidis, a Gram
positive eubacterial species, as the test microorganism (Kamgang
et al., 2007). In both studies, CFU ability was measured prior to
and after treatment and a macroscopic disorganization of the biofilm was observed
using a scanning electron microscopy analysis (Moisan et
al., 2001). As treatment of the biofilm by the non-thermal atmospheric
pressure plasma process can damage the biofilm, it is possible that cell recovery
by scraping and mechanical dispersal of the biofilm-resident cells to measure
CFU ability was affected (Kamgang et al., 2007;
Tryland et al., 1998). Thus, we examined the
potential cell viability using a two-dye system and found that the CFU and staining/fluorescence
microscopy results were concordant.
We have shown that biofilm-resident E. coli, a Gram negative bacterium, are more resistant to non-thermal atmospheric pressure plasma treatment than are planctonic cells. Clearly, further studies are required to determine bacterial resistance mechanisms within biofilms in order to optimize non-thermal atmospheric pressure plasma treatment for surface decontamination.
The authors would like to thank Françoise Le Hégarat and Micheline Terrier for their helpful comments and suggestions. This research was supported by a Plan Pluri-Formations (PPF31-DS8) de lUniversité Paris-Sud 11 and the Centre National de la Recherche Scientifique (CNRS, France).
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